- Email: [email protected]
Detection of 1O2 by EPR techniques
40
Spin Trapping and New Developments in EPR
4.21 5,5-DIMETHYI,-I-PYRROLINE-N-OXIDE ENHANCES THE SPON-
TANEOUS SUP~ROXIDEGEN]~ATION BY PRIMAQUINE Kohji Ichimori, Laszlo Pronai, Hiroe Nakazawa Department of Physiology 2, Tokai University, School of Medicine, Isehara, Kanagaw~, 259-11, Japan Primaquine (PQ), a well known antimalarial drug, has been reported to generate superoxide (02-) with some reducing agents such as NADPH. However, we proved PQ generates 02- without reducing or oxidizing reagents. Furthermore, 5,5'-dimethyl-l-pyrroline-N-oxide (DMPO) enhanced the 02- generation by PQ. Chemiluminescence was detected by adding only PQ to aqueous MCLA, which is a specific chemiluminescent probe for 02- , and completely quenched by superoxide dismutase (SOD); indicating that PQ generates 02 - by itself in aerobic condition. Superoxide spin adducts could be also detected both in aqueous solutions and in dimethyl sulfoxide with DMPO, but not with PBN or POBN, open-chained nitrones, by ESR. The amount of 02- generation showed a linear correlation with DMPO concentration, and SOD inhibited the DMPO-00H formation in a competitive manner, which provided us a simple and inexpensive system for the investigation of superoxide scavenging rate of drugs instead of xanthine oxidase system. The results suggested that PQ is autooxidized in aerobic condition into 5-hydroxy-PQ, which generates 02- , and DMPO accelerates the autooxidation process by trapping 02-. The 5-membered ring nitrones themselves, would enhance the spontaneous 0 - generation by PQ, therefore all studies using t~e ESR/spin trapping technique to explain the mechanism of PQ-related radical generation should be cautiously evaluated.
4.23 DETECTION OF MULTIMODAL COMPLEXATIONBETWEEN
CYCLODEXTRIN AND NITROXIDES BY ESR/ENDOR SPECTROSCOPY Yashige Kotake* and Edward G. Janzen Departments of C l i n i c a l Studies and Biomedical Sciences, The University of Guelph, Guelph, Ontario. NIG 2WI, Canada. Nitroxides (aminoxyls) are known to be incorporated into c y c l o d e x t r i n c a v i t i e s with favorable e q u i l i b r i u m constants. Nitroxides with a second hfsc such as B-hydrogen hfsc provide information much better suited for detailed studies on e q u i l i b r i u m studies of complexation. In addition, i f bulky groups are selected for the n i t r o x i d e probes multimodal complexation can be detected and group e q u i l i b r i u m constants determined. The f e a s i b i l i t y of t h i s type of investigation f o r bimodal complexation has been thoroughly demonstrated f o r n i t r o x i d e s of the f o l l o w i n g type, where C = t e r t - b u t y l , A=phenyl or 2,4,6-trimethoxyphenyl and O, B can be a v a r i e t y of ~CH-N-C d i f f e r e n t groups. Now we also describe a case where trimodal complexation occurs depending on whether the group A, B or C is complexed with B-cyclodextrin. However, the ESR/ENDOR spectroscopy is complicated by the f a c t that both the probe and cyclod e x t r i n are c h i r a l and the complexation produces three pairs of enantiomers and diastereomers. Since ENDOR spectroscopy can provide better res o l u t i o n , four of these have been found %o date with appropriate n i t r o x i d e s . * present address: Oklahoma Medical Research Foundation
FREE RADICAL METABOLISM OF HALOTHANE IN VIVO: DETECTION OF RADICAL ADDUCTS IN BILE Kathryn T. Knecht and Ronald P. Mason Curriculum in Toxicology, UNC, Chapel Hill, NC 27599, U.S.A. and Laboratory of Molecular Biophysics, NIEHS/NIH, Research Triangle Park, NC 27709, U.S.A.
4.22
A free radical metabolite previously detected in the organic extracts of livers from animals treated with halothane and the spin trap c t - p h e n y l - N - t butyl nitrone (PBN) may be related to the hepatoloxicity of halothane. However, this free radical has not been identified and could arise either from metabolism of the parent compound or from lipid peroxidation. In this present study, two radical adduct species have been detected in the bile of living rats treated with halothane and PBN. The treatment of rats with 12% oxygen was required for radical adduct detection. Analysis of the corresponding electron paramagnetic resonance (EPR) spectra obtained when deuterated dI4-PBN was used suggests that these two species result from the spin trapping of a halothanederived (a N = 15.21 G, aH = 4.13 G, and aH= 0.52 G) a~id of a lipid-derived (a ~ = 15.74 G, aI~I ~ 2.10 G, and a T =0.69G(2H))
free radical.
The latter coupling
constants are characteristic of the dl4-PBN/ "CH2R (]3-scission fragment)
radical
adduct.
(This work partially supported by NIH-ES07126)
DETECTION OF 102 BY EPR TECHNIQUES Bibhu R. Misra, Lun-yi Zang and Hara P. Misra D e p a r t m e n t of Biomedical Sciences, Virginia Polytechnic I n s t i t u t e a n d S t a t e University, Blacksburg, VA 24061, U.S.A. The use of TEMP as a detector of 10 2 h a s been p r o p o s e d a n d the p r o d u c t of the TEMp-10 2 reaction h a s b e e n characterized as TEMPO by utilizing EPR techniques (Zang et al. Photochem. Photobiol. in press}. Singlet oxygen w a s g e n e r a t e d during p h o t o i r r a d i a t i o n of various p h o t o s e n s i t i z e r s a n d TEMPO formation was d e t e c t e d by EPR s p e c t r o s c o p y as t h r e e symmetrical hyperfine spllttings with splitting constants aN=16.3 G and a g-value of 2.0056. The rate c o n s t a n t (KT} for the reaction of 10 2 with TEMP to form the TEMPO radical was found to be 5 . 3 x i 0 5 M - l s - l . Azide inhibited the EPR signal intensity in a d o s e - d e p e n d e n t m a n n e r with a q u e n c h i n g rate c o n s t a n t of 3.86x108 M - i s - l . Deuterated solvents augmented the EPR signal intensity. Histidine (3mM), .g-carotene (1.5mM) i n h i b i t e d t h e s i g n a l i n t e n s i t y completely. Superoxide dismutase (0.51~g/ml), ethanol (0.2M), catalase (20~tg/ml) a n d m a n n i t o l (6mM} had trivial e f f e c t s . F u r t h e r m o r e , s u p e r o x i d e g e n e r a t e d e i t h e r via the x a n t h i n e - x a n t h i n e oxidase reaction or from KO2 did not form the TEMPO radical w h e n allowed to react with TEMP. These results indicate that the reaction of converting TEMP to TEMPO requires 102 and can therefore be used as a convenient, specific and sensitive assay for 102 .
4.24
Spin Trapping and New Developments in EPR
4.21 5,5-DIMETHYI,-I-PYRROLINE-N-OXIDE ENHANCES THE SPON-
TANEOUS SUP~ROXIDEGEN]~ATION BY PRIMAQUINE Kohji Ichimori, Laszlo Pronai, Hiroe Nakazawa Department of Physiology 2, Tokai University, School of Medicine, Isehara, Kanagaw~, 259-11, Japan Primaquine (PQ), a well known antimalarial drug, has been reported to generate superoxide (02-) with some reducing agents such as NADPH. However, we proved PQ generates 02- without reducing or oxidizing reagents. Furthermore, 5,5'-dimethyl-l-pyrroline-N-oxide (DMPO) enhanced the 02- generation by PQ. Chemiluminescence was detected by adding only PQ to aqueous MCLA, which is a specific chemiluminescent probe for 02- , and completely quenched by superoxide dismutase (SOD); indicating that PQ generates 02 - by itself in aerobic condition. Superoxide spin adducts could be also detected both in aqueous solutions and in dimethyl sulfoxide with DMPO, but not with PBN or POBN, open-chained nitrones, by ESR. The amount of 02- generation showed a linear correlation with DMPO concentration, and SOD inhibited the DMPO-00H formation in a competitive manner, which provided us a simple and inexpensive system for the investigation of superoxide scavenging rate of drugs instead of xanthine oxidase system. The results suggested that PQ is autooxidized in aerobic condition into 5-hydroxy-PQ, which generates 02- , and DMPO accelerates the autooxidation process by trapping 02-. The 5-membered ring nitrones themselves, would enhance the spontaneous 0 - generation by PQ, therefore all studies using t~e ESR/spin trapping technique to explain the mechanism of PQ-related radical generation should be cautiously evaluated.
4.23 DETECTION OF MULTIMODAL COMPLEXATIONBETWEEN
CYCLODEXTRIN AND NITROXIDES BY ESR/ENDOR SPECTROSCOPY Yashige Kotake* and Edward G. Janzen Departments of C l i n i c a l Studies and Biomedical Sciences, The University of Guelph, Guelph, Ontario. NIG 2WI, Canada. Nitroxides (aminoxyls) are known to be incorporated into c y c l o d e x t r i n c a v i t i e s with favorable e q u i l i b r i u m constants. Nitroxides with a second hfsc such as B-hydrogen hfsc provide information much better suited for detailed studies on e q u i l i b r i u m studies of complexation. In addition, i f bulky groups are selected for the n i t r o x i d e probes multimodal complexation can be detected and group e q u i l i b r i u m constants determined. The f e a s i b i l i t y of t h i s type of investigation f o r bimodal complexation has been thoroughly demonstrated f o r n i t r o x i d e s of the f o l l o w i n g type, where C = t e r t - b u t y l , A=phenyl or 2,4,6-trimethoxyphenyl and O, B can be a v a r i e t y of ~CH-N-C d i f f e r e n t groups. Now we also describe a case where trimodal complexation occurs depending on whether the group A, B or C is complexed with B-cyclodextrin. However, the ESR/ENDOR spectroscopy is complicated by the f a c t that both the probe and cyclod e x t r i n are c h i r a l and the complexation produces three pairs of enantiomers and diastereomers. Since ENDOR spectroscopy can provide better res o l u t i o n , four of these have been found %o date with appropriate n i t r o x i d e s . * present address: Oklahoma Medical Research Foundation
FREE RADICAL METABOLISM OF HALOTHANE IN VIVO: DETECTION OF RADICAL ADDUCTS IN BILE Kathryn T. Knecht and Ronald P. Mason Curriculum in Toxicology, UNC, Chapel Hill, NC 27599, U.S.A. and Laboratory of Molecular Biophysics, NIEHS/NIH, Research Triangle Park, NC 27709, U.S.A.
4.22
A free radical metabolite previously detected in the organic extracts of livers from animals treated with halothane and the spin trap c t - p h e n y l - N - t butyl nitrone (PBN) may be related to the hepatoloxicity of halothane. However, this free radical has not been identified and could arise either from metabolism of the parent compound or from lipid peroxidation. In this present study, two radical adduct species have been detected in the bile of living rats treated with halothane and PBN. The treatment of rats with 12% oxygen was required for radical adduct detection. Analysis of the corresponding electron paramagnetic resonance (EPR) spectra obtained when deuterated dI4-PBN was used suggests that these two species result from the spin trapping of a halothanederived (a N = 15.21 G, aH = 4.13 G, and aH= 0.52 G) a~id of a lipid-derived (a ~ = 15.74 G, aI~I ~ 2.10 G, and a T =0.69G(2H))
free radical.
The latter coupling
constants are characteristic of the dl4-PBN/ "CH2R (]3-scission fragment)
radical
adduct.
(This work partially supported by NIH-ES07126)
DETECTION OF 102 BY EPR TECHNIQUES Bibhu R. Misra, Lun-yi Zang and Hara P. Misra D e p a r t m e n t of Biomedical Sciences, Virginia Polytechnic I n s t i t u t e a n d S t a t e University, Blacksburg, VA 24061, U.S.A. The use of TEMP as a detector of 10 2 h a s been p r o p o s e d a n d the p r o d u c t of the TEMp-10 2 reaction h a s b e e n characterized as TEMPO by utilizing EPR techniques (Zang et al. Photochem. Photobiol. in press}. Singlet oxygen w a s g e n e r a t e d during p h o t o i r r a d i a t i o n of various p h o t o s e n s i t i z e r s a n d TEMPO formation was d e t e c t e d by EPR s p e c t r o s c o p y as t h r e e symmetrical hyperfine spllttings with splitting constants aN=16.3 G and a g-value of 2.0056. The rate c o n s t a n t (KT} for the reaction of 10 2 with TEMP to form the TEMPO radical was found to be 5 . 3 x i 0 5 M - l s - l . Azide inhibited the EPR signal intensity in a d o s e - d e p e n d e n t m a n n e r with a q u e n c h i n g rate c o n s t a n t of 3.86x108 M - i s - l . Deuterated solvents augmented the EPR signal intensity. Histidine (3mM), .g-carotene (1.5mM) i n h i b i t e d t h e s i g n a l i n t e n s i t y completely. Superoxide dismutase (0.51~g/ml), ethanol (0.2M), catalase (20~tg/ml) a n d m a n n i t o l (6mM} had trivial e f f e c t s . F u r t h e r m o r e , s u p e r o x i d e g e n e r a t e d e i t h e r via the x a n t h i n e - x a n t h i n e oxidase reaction or from KO2 did not form the TEMPO radical w h e n allowed to react with TEMP. These results indicate that the reaction of converting TEMP to TEMPO requires 102 and can therefore be used as a convenient, specific and sensitive assay for 102 .
4.24